PROJECT SUMMARY The studies proposed here will for the first time identify the signal transduction mechanisms utilized by tuft cells that orchestrate a type 2 innate lymphoid cell (ILC2) circuit immune response to infectious agents. The R21 mechanism is appropriate because these studies are exploratory and novel in the immune system, employing approaches have not been used previously in this immunity-associated cell-type. The overarching goal of our proposed studies is to explore for the first time the signal-transduction mechanisms and electrophysiological properties of small-intestinal tuft cells. Recent studies have revealed that rare tuft cells are chemosensory sentinels that respond to and orchestrate an immune response to infectious agents including parasitic helminths and protists in the small intestine. Tuft cells release effector molecules including interleukin-25 and acetylcholine to activate a type 2 innate lymphoid cell (ILC2) response. IL-25 activated ILC2 cells release IL-13, which in turn acts on epithelial progenitors to promote expansion of tuft cells and goblet cells, remodeling of the intestine and resolution of the infection. Thus, tuft cells orchestrate a major mucosal immune response, but the signal transduction mechanisms and the electrophysiological properties of these critical cells have not been studied. Small-intestinal tuft cells express key components present in the type II taste-bud cell signal-transduction cascade that responds to sweet, bitter and umami substances, including G protein-coupled receptors (GPCR) (succinate receptor-1 and bitter taste receptor Tas2R), phospholipase C?2 and downstream IP3-mediated Ca2+ release, and the TRPM5 cation channel, leading to the suggestion that the signaling mechanisms are shared between the two cell types. However, evidence for this is lacking. In preliminary experiments, we found that tuft cells lack voltage-gated Na+ currents, have only very small K+ currents and high input resistance, and do not generate action potentials, features quite distinct from those of taste-bud cells. Furthermore, they lack voltage-gated Ca2+ currents, so the sources of Ca2+ required for IL-25 secretion are undefined. We have developed a working model for signal transduction in tuft cells in which an essential role of TRPM5 is promote Na+ influx to raise intracellular Na+ concentration and to strongly depolarize the plasma membrane, thereby activating reverse-mode Na+/Ca2+ exchange to facilitate Ca2+ influx to drive IL- 25 release. This model is supported by our preliminary results that demonstrated that an inhibitor of Na+/Ca2+ exchangers reduced extracellular Ca2+-sensitive currents in single tuft cells and strongly decreased pathogen- induced tuft cell-mediated IL-25 release. The studies proposed here will for the first time identify the signal transduction mechanisms utilized by tuft cells to orchestrate small-intestinal innate immunity, with therapeutic implications. If successful, our insights into the signaling mechanisms of small-intestinal tuft cells will serve as a basis for future studies of tuft-cell and solitary chemosensory-cell signal transduction in other tissues, including airways, olfactory system, gallbladder and thymus.